Haploid induction via unpollinated ovule culture in Gerbera hybrida

Ovule-derived haploid culture is an effective and important method for genetic study and plant breeding. Gerbera hybrida is a highly heterozygous species, and the lack of homozygous lines presents a challenge for molecular genetic research. Therefore, we performed haploid induction through unpollinated ovule culture and evaluated the effects of several important factors on this culturing procedure in G. hybrida, including genotype, low temperature, and the development seasons of the ovules. Among 45 G. hybrida cultivars analyzed, 29 cultivars exhibited adventitious bud induction via in vitro unpollinated ovule culture with significant different responses, indicating that the genotype of donor plants was a vital factor for inducibility. Four cultivars with significantly different induction rates, including one non-induced cultivar, were selected to analyze seasonal effects. Ovules extracted in the summer consistently had the highest induction rates, and even the non-induced cultivar included in the analysis could be induced at low levels when ovules from summer were used. Low temperature treatment could also promote adventitious bud induction, and in particular, a strong and significant effect was detected after 7 days of cold treatment. Ploidy level measurements by flow cytometry revealed that 288 ovule-derived regenerants were haploid (55.17%) and 218 lines were diploid (41.76%). Moreover, genetic stability analysis of the regenerants indicated 100% similarity to the marker profile of the mother plant. This is the first report of ovule-derived haploids in G. hybrida, which may facilitate the development of homozygous lines for molecular research and plant breeding.